Targeted chromosomal mutagenesis using zinc finger nucleases

ABSTRACT

The present invention provides for a method or methods of targeted genetic recombination or mutagenesis in a host cell or organism, and compositions useful for carrying out the method. The targeting method of the present invention exploits endogenous cellular mechanisms for homologous recombination and repair of double stranded breaks in genetic material. The present invention provides numerous improvements over previous mutagenesis methods, such advantages include that the method is generally applicable to a wide variety of organisms, the method is targeted so that the disadvantages associated with random insertion of DNA into host genetic material are eliminated, and certain embodiments require relatively little manipulation of the host genetic material for success. Additionally, it provides a method that produces organisms with specific gene modifications in a short period of time.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application is continuation of U.S. patent application Ser.No. 10/502,565 file on Jul. 22, 2004, which is a National PhaseApplication of International Application No. PCT/US03/002012 filed onJan. 22, 2003, which claims priority from U.S. Provisional PatentApplication No. 60/351,035 filed Jan. 23, 2002, which is herebyincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The U.S. Government has certain rights in the invention based uponpartial support by Grant R01 GM 58504.

BACKGROUND OF THE INVENTION

Gene targeting—the process of gene replacement by homologousrecombination or mutation—is a very useful, but typically inefficienttechnique for introducing desired changes in the genetic material of ahost cell. Only when powerful selection for the targeted product can beapplied is recovery of the desired alteration possible. A general methodfor improving the efficiency of gene targeting would be valuable in manycircumstances, as would extension of this tool to a broader range oforganisms.

It has been demonstrated in model experiments that introduction of adouble-strand break (DSB) in host DNA greatly enhances the frequency oflocalized recombination. However, those tests required insertion of arecognition site for a specific endonuclease before cleavage could beinduced. Similarly, in Drosophila the DSBs produced by P-elementexcision are recombinagenic, but require the P-element to preexist atthe target site.

Although previously demonstrated methods of genetic transformation hadbeen highly successful, transformation without targeted recombinationhas also been accompanied by problems associated with random insertionof the introduced DNA. Random integration can lead to the inactivationof essential genes, or to the aberrant expression of the introducedgene. Additional problems associated with genetic transformation includemosaicism due to multiple integrations, and technical difficultiesassociated with generation of replication defective recombinant viralvectors.

Targeted genetic recombination or mutation of a cell or organism is nowpossible because complete genomic sequences have been determined for anumber of organisms, and more sequences are being obtained each day. Notonly would the ability to direct a mutation to a specific genetic locusgreatly aid those studying the function of particular genes, targetedgenetic recombination would also have therapeutic and agriculturalapplications. Methods of targeted genetic recombination are needed thatare more general, efficient, and/or reproducible than currentlyavailable techniques.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for carrying outtargeted genetic recombination or mutation. Any segment of endogenousnucleic acid in a cell or organism can be modified by the method of theinvention as long as the sequence of the target region, or portion ofthe target region, is known, or if isolated DNA homologous to the targetregion is available.

In certain embodiments, the compositions and methods comprise thetransformation of a host organism by introducing a nucleic acid moleculeencoding a chimeric zinc finger nuclease into a cell or organism andidentifying a resulting cell or organism in which a selected endogenousDNA sequence is cleaved and exhibits a mutation.

In a preferred embodiment, such methods comprise selecting a zinc fingerDNA binding domain capable of preferentially binding to a specific hostDNA locus to be mutated; further selecting a non-specific DNA cleavagedomain capable of cleaving double-stranded DNA when operatively linkedto said binding domain and introduced into the host cell; furtherselecting an inducible promoter region capable of inducing expression inthe host cell; and further operatively linking DNA encoding the bindingdomain and the cleavage domain and the inducible promoter region toproduce a DNA construct. The DNA construct is then introduced into atarget host cell and at least one host cell exhibiting recombination atthe target locus in the host DNA is identified. In a particularembodiment, the DNA binding domain comprises the binding domains ofthree Cis₂His₂ zinc fingers. In another embodiment, the cleavage domaincomprises a cleavage domain derived from the Type II restrictionendonuclease FokI. In one embodiment, an inducible heat shock promoteris operatively linked to DNA encoding the chimeric zinc finger nuclease.

Additional embodiments involve methods for targeted insertion byhomologous recombination of selected DNA sequences (donor DNA). DonorDNA can comprise a sequence that encodes a product to be produced in thehost cell. Said product can be a product produced for the benefit of thehost cell or organism (for example, gene therapy), or the product can beone that is produced for use outside the host cell or organism (forexample, the product may be selected from, but not limited to,pharmaceuticals, hormones, protein products used in the manufacture ofuseful objects or devices, nutriceuticals, products used in chemicalmanufacture or synthesis, etc.).

In a certain embodiment, the present invention is utilized to disrupt atargeted gene in a somatic cell. Such gene may be over-expressed in oneor more cell types resulting in disease. Disruption of such gene mayonly be successful in a low percentage of somatic cells but suchdisruption may contribute to better health for an individual sufferingfrom disease due to over-expression of such gene.

In another embodiment, the present invention can be utilized to disrupta targeted gene in a germ cell. Cells with such disruption in thetargeted gene can be selected for in order to create an organism withoutfunction of the targeted gene. In such cell the targeted gene functioncan be completely knocked out.

In another embodiment, the present invention can be utilized to enhanceexpression of a particular gene by the insertion of a control elementinto a somatic cell. Such a control element may be selected from a groupconsisting of, but not limited to, a constitutively active, inducible,tissue-specific or development stage-specific promoters. Such controlelement may be targeted to a chromosomal locus where it will effectexpression of a particular gene that is responsible for a product with atherapeutic effect in such a cell or the host organism. The presentinvention may further provide for the insertion of donor DNA containinga gene encoding a product that, when expressed, has a therapeutic effecton the host cell or organism. An example of such a therapeutic methodwould be to use the targeted genetic recombination of the presentinvention to effect insertion into a pancreatic cell of an activepromoter operatively linked to donor DNA containing an insulin gene. Thepancreatic cell containing the donor DNA would then produce insulin,thereby aiding a diabetic host. Additionally, donor DNA constructs couldbe inserted into a crop genome in order to effect the production of apharmaceutical relevant gene product. A gene encoding a pharmaceuticaluseful protein product, such as insulin or hemoglobin, functionallylinked to a control element, such as a constitutively active, inducible,tissue-specific or development stage-specific promoters, could beinserted into a host plant in order to produce a large amount of thepharmaceutically useful protein product in the host plant. Such proteinproducts could then be isolated from the plant. Alternatively, theabove-mentioned methods can be utilized in a germ cell.

The present invention can be utilized in both somatic and germ linecells to effect alteration at any chromosomal target locus.

Methods of the present invention are applicable to a wide range of celltypes and organisms. The present invention can apply to any of thefollowing cells, although the methods of the invention are not limitedto the cells or organisms herein listed: A single celled ormulticellular organism; an oocyte; a gamete; a germline cell in cultureor in the host organism; a somatic cell in culture or in the hostorganism; an insect cell, including an insect selected from the groupconsisting of Coleoptera, Diptera, Hemiptera, Homoptera, Hymenoptera,Lepidoptera, or Orthoptera, including a fruit fly, a mosquito and amedfly; a plant cell, including a monocotyledon cell and a dicotyledoncell; a mammalian cell, including but not limited to a cell selectedfrom the group consisting of mouse, rat, pig, sheep, cow, dog or catcells; an avian cell, including, but not limited to a cell selected fromthe group consisting of chicken, turkey, duck or goose cells; or a fishcell, including, but not limited to zebrafish, trout or salmon cells.

Many alterations and variations of the invention exist as describedherein. The invention is exemplified for targeted genetic recombinationin the insect, Drosophila and the plant, Arabidopsis. In Drosophila andArabidopsis, the nucleotide sequence is known for most of the genome.Large segments of genomic sequences from other organisms are becomingknown at a fast pace. The elements necessary to carry out the methods ofthe present invention as herein disclosed can be adapted for applicationin any cell or organism. The invention therefore provides a generalmethod for targeted genetic recombination in any cell or organism.

Table 1: Illustrates the number of germline mutants recovered bycrossing males exposed to a heat shock with attached-X [C(1)DX] femalesand females from the heat shock to FM6 (y) males in accordance with anembodiment of the present invention. The percent of all the heat-shockedparents screened that gave at least one germline mutant is shown inparentheses in the # Giving y column. The total number of mutant fliesrecovered is given in the Total y column and also expressed as a percentof all candidate offspring (in parentheses). The number of mutantoffspring per fly varied from 1 to 15. The ND data are from M. Bibikovaet al. (2002) Genetics 161: 1169-1175 which is hereby incorporated byreference.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for carryingout targeted genetic recombination or mutation. In contrast topreviously known methods for targeted genetic recombination, the presentinvention is efficient and inexpensive to perform and is adaptable toany cell or organism. Any segment of double-stranded nucleic acid of acell or organism can be modified by the method of the present invention.The method exploits both homologous and non-homologous recombinationprocesses that are endogenous in all cells.

The method of the present invention provides for both targeted DNAinsertions and targeted DNA deletions. The method involvestransformation of a cell with a nucleic acid construct minimallycomprising DNA encoding a chimeric zinc finger nuclease (ZFN). In aparticular embodiment, the method further involves transforming a cellwith a nucleic acid construct comprising donor DNA. Other schemes basedon these general concepts are within the scope and spirit of theinvention, and are readily apparent to those skilled in the art.

The present invention can be utilized in both somatic and germ cells toconduct genetic manipulation at a particular genetic locus.

In a particular embodiment, the present invention is utilized to disrupta gene in a somatic cell wherein that gene is over-expressing a productand/or expressing a product that is deleterious to the cell or organism.Such gene may be over-expressed in one or more cell types resulting indisease. Disruption of such gene by the methods of the present inventionmay contribute to better health for an individual suffering from diseasedue to expression of such gene. In other words, disruption of genes ineven a small percentage of cells can work to decrease expression levelsin order to produce a therapeutic effect.

In another embodiment, the present invention can be utilized to disrupta gene in a germ cell. Cells with such disruption in a particular genecan be selected for in order to create an organism without function ofsuch gene. In such cell the gene can be completely knocked-out. Theabsence of function in this particular cell can have a therapeuticeffect.

In another embodiment, the present invention can be utilized to enhanceexpression of a particular gene by the insertion of a control elementinto a somatic cell. Such control element may be a constitutivelyactive, inducible or development stage-specific promoter. It may also bea tissue-specific promoter capable of effecting expression only inparticular cell types. Such control element may be placed in such amanner to effect expression of a particular gene that is responsible fora product with a therapeutic effect in such a cell.

The present invention may further provide for the insertion of donor DNAencoding a gene product that, when constitutively expressed, has atherapeutic effect. An example of this embodiment would be to insertsuch DNA constructs into an individual suffering from diabetes in orderto effect insertion of an active promoter and donor DNA encoding theinsulin gene in a population of pancreatic cells. This population ofpancreatic cells containing the exogenous DNA would then produceinsulin, thereby aiding the diabetic patient. Additionally, such DNAconstructs could be inserted into crops in order to effect theproduction of pharmaceutically-relevant gene products. Genes for proteinproducts, such as insulin, lipase or hemoglobin, could be inserted intoplants along with control elements, such as constitutively active orinducible promoters, in order to produce large amounts of thesepharmaceuticals in a plant. Such protein products could then be isolatedfrom the plant. Transgenic plants or animals may be produced with thismethod through a nuclear transfer technique (McCreath, K. J. et al.(2000) Nature 405: 1066-1069; Polejaeva, I. A. et al., (2000) Nature407: 86-90). Tissue or cell-type specific vectors may also be employedfor providing gene expression only in the cells of choice.

Alternatively, the above-mentioned methods can be utilized in a germcell in order to select cells where insertion has occurred in theplanned manner in order for all subsequent cell divisions to producecells with the desired genetic change.

As used herein, the cells in which genetic manipulation occurs and anexogenous DNA segment or gene has been introduced through the hand ofman are called recombinant cells. Therefore, recombinant cells aredistinguishable from naturally occurring cells which do not contain arecombinantly introduced exogenous DNA segment or gene. Recombinantcells include those having an introduced cDNA or genomic gene, and alsoinclude genes positioned adjacent to a heterologous promoter notnaturally associated with the particular introduced gene.

To express a recombinant encoded protein or peptide, whether mutant orwild-type, in accordance with the present invention one would prepare anexpression vector that comprises isolated nucleic acids under thecontrol of, or operatively linked to, one or more promoters, which maybe inducible, constitutively active or tissue specific, for example. Tobring a coding sequence “under the control of a promoter, one positionsthe 5′ end of the transcription initiation site of the transcriptionalreading frame generally between about 1 and about 50 nucleotides“downstream” (i.e., 3′) of the chosen promoter. The “upstream” promoterstimulates transcription of the DNA and promotes expression of theencoded recombinant protein. This is the meaning of “recombinantexpression” in this context.

Ways of effecting protein expression are well known in the art. Oneskilled in the art is capable of expression a protein of his or herchoice in accordance with the present invention.

The methods of the present invention can be applied to whole organismsor in cultured cells or tissues or nuclei, including those cells,tissues or nuclei that can be used to regenerate an intact organism, orin gametes such as eggs or sperm in varying stages of their development.Because DSBs stimulate mutagenic repair in essentially all cells ororganisms, cleavage by ZFNs may be used in any cells or organisms. Themethods of the present invention can be applied to cells derived anyorganism, including but not limited to insects, fungi, rodents, cows,sheep, goats, chickens, and other agriculturally important animals, aswell as other mammals, including, but not limited to dogs, cats andhumans.

Additionally, the compositions and methods of the present invention maybe used in plants. It is contemplated that the compositions and methodscan be used in any variety of plant species, such as monocots or dicots.In certain embodiments, the invention can be used in plants such asgrasses, legumes, starchy staples, Brassica family members, herbs andspices, oil crops, ornamentals, woods and fibers, fruits, medicinalplants, poisonous plants, corn, cotton, castor bean and any other cropspecie. In alternative embodiments, the invention can be used in plantssuch as sugar cane, wheat, rice, maize, potato, sugar beet, cassava,barley, soybean, sweet potato, oil palm fruit, tomato, sorghum, orange,grape, banana, apple, cabbage, watermelon, coconut, onion, cottonseed,rapeseed and yam. In some embodiments, the invention can be used inmembers of the Solanaceae specie, such as tobacco, tomato, potato andpepper. In other embodiments, the invention can be used in poisonousornamentals, such as oleander, any yew specie and rhododendron. In aparticular embodiment, the Brassica specie is Arabidopsis.

Grasses include, but are not limited to, wheat, maize, rice, rye,triticale, oats, barley, sorghum, millets, sugar cane, lawn grasses andforage grasses. Forage grasses include, but are not limited to, Kentuckybluegrass, timothy grass, fescues, big bluestem, little bluestem andblue gamma. Legumes include, but are not limited to, beans like soybean,broad or Windsor bean, kidney bean, lima bean, pinto bean, navy bean,wax bean, green bean, butter bean and mung bean; peas like green pea,split pea, black-eyed pea, chick-pea, lentils and snow pea; peanuts;other legumes like carob, fenugreek, kudzu, indigo, licorice, mesquite,copaifera, rosewood, rosary pea, senna pods, tamarind, and tuba-root;and forage crops like alfalfa. Starchy staples include, but are notlimited to, potatoes of any species including white potato, sweetpotato, cassava, and yams. Brassica, include, but are not limited to,cabbage, broccoli, cauliflower, brussel sprouts, turnips, collards, kaleand radishes. Oil crops include, but are not limited to, soybean, palm,rapeseed, sunflower, peanut, cottonseed, coconut, olive palm kernel.Woods and fibers include, but are not limited to, cotton, flax, andbamboo. Other crops include, but are not limited to, quinoa, amaranth,tarwi, tamarillo, oca, coffee, tea, and cacao.

DEFINITIONS

For the purposes of the present invention, the following terms shallhave the following meanings:

As used herein, the term “targeted genetic recombination” refers to aprocess wherein recombination occurs within a DNA target locus presentin a host cell or host organism. Recombination can involve eitherhomologous or non-homologous DNA. One example of homologous targetedgenetic recombination would be cleavage of a selected locus of host DNAby a zinc finger nuclease (ZFN), followed by homologous recombination ofthe cleaved DNA with homologous DNA of either exogenous or endogenousorigin. One example of non-homologous targeted genetic recombinationwould be cleavage of a selected locus of host DNA by a ZFN, followed bynon-homologous end joining (NHEJ) of the cleaved DNA.

As used herein, the terms “host cell” or “host organism” or, simply,“target host”, refer to a cell or an organism that has been selected tobe genetically transformed to carry one or more genes for expression ofa function used in the methods of the present invention. A host canfurther be an organism or cell that has been transformed by the targetedgenetic recombination or mutation methods of the present invention.

The term “target” or “target locus” or “target region” refers herein tothe gene or DNA segment selected for modification by the targetedgenetic recombination method of the present invention. Ordinarily, thetarget is an endogenous gene, coding segment, control region, intron,exon or portion thereof, of the host organism. However, the target canbe any part or parts of the host DNA.

For the purposes of the present invention, the term “zinc fingernuclease” or “ZFN” refers to a chimeric protein molecule comprising atleast one zinc finger DNA binding domain effectively linked to at leastone nuclease capable of cleaving DNA. Ordinarily, cleavage by a ZFN at atarget locus results in a double stranded break (DSB) at that locus.

For the purposes of the present invention, the term “marker” refers to agene or sequence whose presence or absence conveys a detectablephenotype to the host cell or organism. Various types of markersinclude, but are not limited to, selection markers, screening markersand molecular markers. Selection markers are usually genes that can beexpressed to convey a phenotype that makes an organism resistant orsusceptible to a specific set of environmental conditions. Screeningmarkers can also convey a phenotype that is a readily observable anddistinguishable trait, such as Green Fluorescent Protein (GFP), GUS orbeta-galactosidase. Molecular markers are, for example, sequencefeatures that can be uniquely identified by oligonucleotide probing, forexample RFLP (restriction fragment length polymorphism), or SSR markers(simple sequence repeat).

As used herein, the term “donor” or “donor construct” refers to theentire set of DNA segments to be introduced into the host cell ororganism as a functional group. The term “donor DNA” as used hereinrefers to a DNA segment with sufficient homology to the region of thetarget locus to allow participation in homologous recombination at thesite of the targeted DSB.

For the purposes of the present invention, the term “gene” refers to anucleic acid sequence that includes the translated sequences that encodea protein (“exons”), the untranslated intervening sequences (“introns”),the 5′ and 3′ untranslated region and any associated regulatoryelements.

For the purposes of the present invention, the term “sequence” means anyseries of nucleic acid bases or amino acid residue, and may or may notrefer to a sequence that encodes or denotes a gene or a protein. Many ofthe genetic constructs used herein are described in terms of therelative positions of the various genetic elements to each other. Forthe purposes of the present invention, the term “adjacent” is used toindicate two elements that are next to one another without implyingactual fusion of the two elements. Additionally, for the purposes of thepresent invention, “flanking” is used to indicate that the same,similar, or related sequences exist on either side of a given sequence.Segments described as “flanking” are not necessarily directly fused tothe segment they flank, as there can be intervening, non-specified DNAbetween a given sequence and its flanking sequences. These and otherterms used to describe relative position are used according to normalaccepted usage in the field of genetics.

For the purposes of the present invention, the term “recombination,” isused to indicate the process by which genetic material at a given locusis modified as a consequence of an interaction with other geneticmaterial. For the purposes of the present invention, the term“homologous recombination” is used to indicate recombination occurringas a consequence of interaction between segments of genetic materialthat are homologous, or identical. In contrast, for purposes of thepresent invention, the term “non-homologous recombination” is used toindicate a recombination occurring as a consequence of interactionbetween segments of genetic material that are not homologous, oridentical. Non-homologous end joining (NHEJ) is an example ofnon-homologous recombination.

Moreover, for the purposes of the present invention, the term “a” or“an” entity refers to one or more than one of that entity; for example,“a protein” or “an nucleic acid molecule” refers to one or more of thosecompounds, or at least one compound. As such, the terms “a” or “an”,“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising,” “including,” and“having” can be used interchangeably. Furthermore, a compound “selectedfrom the group consisting of refers to one or more of the compounds inthe list that follows, including mixtures (i.e. combinations) of two ormore of the compounds. According to the present invention, an isolatedor biologically pure compound is a compound that has been removed fromits natural milieu. As such, “isolated” and “biologically pure” do notnecessarily reflect the extent to which the compound has been purified.An isolated compound of the present invention can be obtained from itsnatural source, can be produced using molecular biology techniques orcan be produced by chemical synthesis.

Zinc Finger Nucleases

A zinc finger nuclease (ZFN) of the present invention is a chimericprotein molecule capable of directing targeted genetic recombination ortargeted mutation in a host cell by causing a double stranded break(DSB) at the target locus. A ZFN of the present invention includes aDNA-binding domain and a DNA-cleavage domain, wherein the DNA bindingdomain is comprised of at least one zinc finger and is operativelylinked to a DNA-cleavage domain. The zinc finger DNA-binding domain isat the N-terminus of the chimeric protein molecule and the DNA-cleavagedomain is located at the C-terminus of said molecule.

A ZFN as herein described must have at least one zinc finger. In apreferred embodiment a ZFN of the present invention would have at leastthree zinc fingers in order to have sufficient specificity to be usefulfor targeted genetic recombination in a host cell or organism. A ZFNcomprising more than three zinc fingers is within the scope of theinvention. A ZFN having more than three zinc fingers, although moretime-consuming to construct, would have progressively greaterspecificity with each additional zinc finger. In a particularembodiment, the DNA-binding domain is comprised of three zinc fingerpeptides operatively linked to a DNA cleavage domain.

The zinc finger domain of the present invention can be derived from anyclass or type of zinc finger. In a particular embodiment, the zincfinger domain comprises the Cis₂His₂ type of zinc finger that is verygenerally represented, for example, by the zinc finger transcriptionfactors TFIIIA or Sp1. In a preferred embodiment, the zinc finger domaincomprises three Cis₂His₂ type zinc fingers. The DNA recognition and/orthe binding specificity of a ZFN can be altered in order to accomplishtargeted genetic recombination at any chosen site in cellular DNA. Suchmodification can be accomplished using known molecular biology and/orchemical synthesis techniques. (see, for example, M. Bibikova et al.(2002) Genetics 161: 1169-1175). ZFNs comprising zinc fingers having awide variety of DNA recognition and/or binding specificities are withinthe scope of the present invention.

The ZFN DNA-cleavage domain is derived from a class of non-specific DNAcleavage domains, for example the DNA-cleavage domain of a Type IIrestriction enzyme. In a particular embodiment the DNA-cleavage domainis derived from the Type 11 restriction enzyme, FokI.

In a preferred embodiment, a ZFN comprises three Cis₂His₂ type of zincfingers, and a DNA-cleavage domain derived from the type II restrictionenzyme, FokI According to this preferred embodiment, each zinc fingercontacts 3 consecutive base pairs of DNA creating a 9 bp recognitionsequence for the ZFN DNA binding domain. The DNA-cleavage domain of thepreferred embodiment requires dimerization of two ZFN DNA-cleavagedomains for effective cleavage of double-stranded DNA. (See, forexample, J. Smith et al., (2000) Nucleic Acids Res. 28: 3361-3369). Thisimposes a requirement for two inverted recognition (target DNA) siteswithin close proximity for effective targeted genetic recombination. Ifall positions in the target sites are contacted specifically, theserequirements enforce recognition of a total of 18 base pairs of DNA.There may be a space between the two sites. The space betweenrecognition sites for ZFNs of the present invention may be equivalent to6 to 35 bp of DNA. The region of DNA between the two recognitions sitesis herein referred to as the “spacer”.

A linker, if present, between the cleavage and recognition domains ofthe ZFN comprises a sequence of amino acid residues selected so that theresulting linker is flexible. Or, for maximum target site specificity,linkerless constructs are made. A linkerless construct has a strongpreference for binding to and then cleaving between recognition sitesthat are 6 bp apart. However, with linker lengths of between 0 and 18amino acids in length, ZFN-mediated cleavage occurs between recognitionsites that are between 5 and 35 bp apart. For a given linker length,there will be a limit to the distance between recognition sites that isconsistent with both binding and dimerization. (M. Bibikova et al.(2001) Mol. Cell. Biol. 21: 289-287). In a preferred embodiment, thereis no linker between the cleavage and recognition domains, and thetarget locus comprises two nine nucleotide recognition sites in invertedorientation with respect to one another, separated by a six nucleotidespacer.

In order to target genetic recombination or mutation according to apreferred embodiment of the present invention, two 9 bp zinc finger DNArecognition sequences must be identified in the host DNA. Theserecognition sites will be in an inverted orientation with respect to oneanother and separated by about 6 bp of DNA. ZFNs are then generated bydesigning and producing zinc finger combinations that bind DNAspecifically at the target locus, and then linking the zinc fingers to acleavage domain of a Type II restriction enzyme.

Targeted Genetic Recombination or Mutation

The method of the present invention can be used for targeted geneticrecombination or mutation of any cell or organism. Minimum requirementsinclude a method to introduce genetic material into a cell or organism(either stable or transient transformation), sequence informationregarding the endogenous target region, and a ZFN construct orconstructs that recognizes and cleaves the target locus. According tosome applications of the present invention, for example homologousrecombination, donor DNA may also be required.

According to another application of the present invention, DNA encodingan identifiable marker will also be included with the DNA construct.Such markers may include a gene or sequence whose presence or absenceconveys a detectable phenotype to the host cell or organism. Varioustypes of markers include, but are not limited to, selection markers,screening markers and molecular markers. Selection markers are usuallygenes that can be expressed to convey a phenotype that makes an organismresistant or susceptible to a specific set of environmental conditions.Screening markers can also convey a phenotype that is a readilyobservable and distinguishable trait, such as Green Fluorescent Protein(GFP), beta-glucuronidase (GUS) or beta-galactosidase. Markers may alsobe negative or positive selectable markers. In a particular embodiment,such negative selectable marker is codA. Molecular markers are, forexample, sequence features that can be uniquely identified byoligonucleotide probing, for example RFLP (restriction fragment lengthpolymorphism), or SSR markers (simple sequence repeat).

The efficiency with which endogenous homologous recombination occurs inthe cells of a given host varies from one class of cell or organism toanother. However the use of an efficient selection method or a sensitivescreening method can compensate for a low rate of recombination.Therefore, the basic tools for practicing the invention are available tothose of ordinary skill in the art for a wide range and diversity ofcells or organisms such that the successful application of such tools toany given host cell or organism is readily predictable. The compositionsand methods of the present invention can be designed to introduce atargeted mutation or genetic recombination into any host cell ororganism. The flexibility of the present invention allows for geneticmanipulation in order to create genetic models of disease or toinvestigate gene function.

The compositions and methods of the present invention can also be usedto effect targeted genetic recombination or mutation in a mammaliancell. In addition, a ZFN can be designed to cleave a particular gene orchromosomal locus, which is then injected into an isolated embryo priorto reimplantation into a female. ZFN-mediated DNA cleavage can occureither in the presence or absence of donor DNA. Off-springs can then bescreened for the desired genetic alteration.

The compositions and methods of the present invention can also be usedaccomplish germline gene therapy in mammals. In one embodiment, ZFNscould be designed to target particular genes of interest. Eggs and spermcould be collected and in-vitro fertilization performed. At the zygotestage, the embryo could be treated with both a ZFN designed to target aparticular sequence and a donor DNA segment carrying a sequence withoutthe deleterious mutation. The embryo could then be returned to a femaleor a uterine alternative for the rest of the gestational period. In aparticular embodiment, for example, the deleterious gene is the commoncystic fibrosis (CF) allele delta F508. ZFNs and donor DNA are usedaccording to the methods of the present invention in order to alleviatedisease caused by a mutant gene. According to the method, eggs and spermfrom known carrier parents are collected and in-vitro fertilized. Afterin-vitro fertilization, the zygote could be injected with ZFNs designedto target the delta F508 allele, and with donor DNA carrying thewild-type allele. The transformed zygote could then be reimplanted intothe mother. With the compositions and methods of the present invention,such gene replacement would allow the offspring and all descendants tobe free of the CF mutation.

In another embodiment, homologous recombination can be used as follows.First, a site for integration is selected within the host cell.Sequences homologous to the integration site are then included in agenetic construct, flanking the selected gene to be integrated into thegenome. Flanking, in this context, simply means that target homologoussequences are located both upstream (5′) and downstream (3′) of theselected gene. These sequences should correspond to some sequencesupstream and downstream of the target gene. The construct is thenintroduced into the cell, thus permitting recombination between thecellular sequences and the construct.

As a practical matter, the genetic construct will normally act as farmore than a vehicle to insert the gene into the genome. For example, itis important to be able to select for recombinants and, therefore, it iscommon to include within the construct a selectable marker gene. Themarker permits selection of cells that have integrated the constructinto their genomic DNA. In addition, homologous recombination may beused to “knock-out” (delete) or interrupt a particular gene. Thus,another approach for inhibiting gene expression involves the use ofhomologous recombination, or “knockout technology”. This is accomplishedby including a mutated or vastly deleted form of the heterologous genebetween the flanking regions within the construct. Thus, it is possible,in a single recombinational event, to (i) “knock out” an endogenousgene, (ii) provide a selectable marker for identifying such an event and(iii) introduce a transgene for expression.

The frequency of homologous recombination in any given cell isinfluenced by a number of factors. Different cells or organisms varywith respect to the amount of homologous recombination that occurs intheir cells and the relative proportion of homologous recombination thatoccurs is also species-variable. The length of the region of homologybetween donor and target affects the frequency of homologousrecombination events, the longer the region of homology, the greater thefrequency. The length of the region of homology needed to observehomologous recombination is also species specific. However, differencesin the frequency of homologous recombination events can be offset by thesensitivity of selection for the recombinations that do occur. It willbe appreciated that absolute limits for the length of the donor-targethomology or for the degree of donor-target homology cannot be fixed butdepend on the number of potential events that can be scored and thesensitivity of the selection for homologous recombination events. Whereit is possible to screen 10⁹ events, for example, in cultured cells, aselection that can identify 1 recombination in 10⁹ cells will yielduseful results. Where the organism is larger, or has a longer generationtime, such that only 100 individuals can be scored in a single test, therecombination frequency must be higher and selection sensitivity is lesscritical.

The method of the present invention dramatically increases theefficiency of homologous recombination in the presence ofextrachromosomal donor DNA (see Examples). The invention can be mostreadily carried out in the case of cells or organisms that have rapidgeneration times or for which sensitive selection systems are available,or for organisms that are single-celled or for which pluripotent celllines exist that can be grown in culture and which can be regenerated orincorporated into adult organisms. Rapid generation time is theadvantage demonstrated for the fruit fly, Drosophila, in the presentinvention. The plant cells, Arabidopsis are one example of pluripotentcells that can be grown in culture then regenerated or incorporated intoan intact organism. These cells or organisms are representative of theirrespective classes and the description demonstrates how the inventioncan be applied throughout those classes. It will be understood by thoseskilled in the art that the invention is operative independent of themethod used to transform the organism. Further, the fact that theinvention is applicable to such disparate organisms as plants andinsects demonstrates the widespread applicability of the invention toliving organisms generally.

Nucleic Acid Delivery

Transformation can be carried out by a variety of known techniques whichdepend on the particular requirements of each cell or organism. Suchtechniques have been worked out for a number of organisms and cells, andcan be adapted without undue experimentation to all other cells. Stabletransformation involves DNA entry into cells and into the cell nucleus.For single-celled organisms and organisms that can be regenerated fromsingle-cells (which includes all plants and some mammals),transformation can be carried out in in vitro culture, followed byselection for transformants and regeneration of the transformants.Methods often used for transferring DNA or RNA into cells includeforming DNA or RNA complexes with cationic lipids, liposomes or othercarrier materials, micro-injection, particle gun bombardment,electroporation, and incorporating transforming DNA or RNA into virusvectors. Other techniques are well known in the art.

Examples of Some Delivery Systems Useful in Practicing the PresentInvention

Liposomal Formulations:

In certain broad embodiments of the invention, the oligo- orpolynucleotides and/or expression vectors containing ZFNs and, whereappropriate, donor DNA, may be entrapped in a liposome. Liposomes arevesicular structures characterized by a phospholipid bilayer membraneand an inner aqueous medium. Multilamellar liposomes have multiple lipidlayers separated by aqueous medium. They form spontaneously whenphospholipids are suspended in an excess of aqueous solution. The lipidcomponents undergo self-rearrangement before the formation of closedstructures and entrap water and dissolved solutes between the lipidbilayer. Also contemplated are cationic lipid-nucleic acid complexes,such as lipofectamine-nucleic acid complexes. Lipids suitable for useaccording to the present invention can be obtained from commercialsources. Liposomes used according to the present invention can be madeby different methods and such methods are known in the art. The size ofthe liposomes varies depending on the method of synthesis.

Microinjection:

Direct microinjection of DNA into various cells, including egg or embryocells, has also been employed effectively for transforming many species.In the mouse, the existence of pluripotent embryonic stem (ES) cellsthat are culturable in vitro has been exploited to generate transformedmice. The ES cells can be transformed in culture, then micro-injectedinto mouse blastocysts, where they integrate into the developing embryoand ultimately generate germline chimeras. By interbreeding heterozygoussiblings, homozygous animals carrying the desired gene can be obtained.

Adenoviruses:

Human adenoviruses are double-stranded DNA tumor viruses with genomesizes of approximate 36 Kb. As a model system for eukaryotic geneexpression, adenoviruses have been widely studied and wellcharacterized, which makes them an attractive system for development ofadenovirus as a gene transfer system. This group of viruses is easy togrow and manipulate, and they exhibit a broad host range in vitro and invivo. In lyrically infected cells, adenoviruses are capable of shuttingoff host protein synthesis, directing cellular machineries to synthesizelarge quantities of viral proteins, and producing copious amounts ofvirus.

Particular advantages of an adenovirus system for delivering DNAencoding foreign proteins to a cell include (i) the ability tosubstitute relatively large pieces of viral DNA with foreign DNA; (ii)the structural stability of recombinant adenoviruses; (iii) the safetyof adenoviral administration to humans; and (iv) lack of any knownassociation of adenoviral infection with cancer or malignancies; (v) theability to obtain high titers of recombinant virus; and (vi) the highinfectivity of adenovirus.

In general, adenovirus gene transfer systems are based upon recombinant,engineered adenovirus which is rendered replication-incompetent bydeletion of a portion of its genome, such as E1, and yet still retainsits competency for infection. Sequences encoding relatively largeforeign proteins can be expressed when additional deletions are made inthe adenovirus genome. For example, adenoviruses deleted in both the E1and E3 regions are capable of carrying up to 10 kB of foreign DNA andcan be grown to high titers in 293 cells.

Other Viral Vectors as Expression Constructs.

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from, for example, vaccinia virus,adeno-associated virus (AAV), and herpes viruses may be employed.Defective hepatitis B viruses, may be used for transformation of hostcells. In vitro studies show that the virus can retain the ability forhelper-dependent packaging and reverse transcription despite thedeletion of up to 80% of its genome. Potentially large portions of theviral genome can be replaced with foreign genetic material. Thehepatotropism and persistence (integration) are particularly attractiveproperties for liver-directed gene transfer. The chloramphenicolacetyltransferase (CAT) gene has been successfully introduced into duckhepatitis B virus genome in the place of the viral polymerase, surface,and pre-surface coding sequences. The defective virus was cotransfectedwith wild-type virus into an avian hepatoma cell line, and culture mediacontaining high titers of the recombinant virus were used to infectprimary duckling hepatocytes. Stable CAT gene expression wassubsequently detected.

Non-Viral Methods.

Several non-viral methods are contemplated by the present invention forthe transfer into a host cell of DNA constructs encoding ZFNs and, whenappropriate, donor DNA. These include calcium phosphate precipitation,lipofectamine-DNA complexes, and receptor-mediated transfection. Some ofthese techniques may be successfully adapted for in vivo or ex vivo use.

In one embodiment of the invention, the expression construct may simplyconsist of naked recombinant DNA. Transfer of the construct may beperformed by any of the DNA transfer methods mentioned above whichphysically or chemically permeabilize the cell membrane. For example,polyomavirus DNA in the form of CaPO4 precipitates was successfullyinjected into liver and spleen of adult and newborn mice which thendemonstrated active viral replication and acute infection. In addition,direct intraperitoneal injection of CaPO4 precipitated plasmidexpression vectors results in expression of the transfected genes.

Transformation of Plants:

Transformed plants are obtained by a process of transforming wholeplants, or by transforming single cells or tissue samples in culture andregenerating whole plants from the transformed cells. When germ cells orseeds are transformed there is no need to regenerate whole plants, sincethe transformed plants can be grown directly from seed. A transgenicplant can be produced by any means known in the art, including but notlimited to Agrobacterium tumefaciens-mediated DNA transfer, preferablywith a disarmed T-DNA vector, electroporation, direct DNA transfer, andparticle bombardment. Techniques are well-known to the art for theintroduction of DNA into monocots as well as dicots, as are thetechniques for culturing such plant tissues and regenerating thosetissues. Regeneration of whole transformed plants from transformed cellsor tissue has been accomplished in most plant genera, both monocots anddicots, including all agronomically important crops.

Screening for Mutations

Methods for genetic screening to accurately detect mutations in genomicDNA, cDNA or RNA samples may be employed, depending on the specificsituation. A number of different methods have been used to detect pointmutations, including denaturing gradient gel electrophoresis (“DGGE”),restriction enzyme polymorphism analysis, chemical and enzymaticcleavage methods, and others. The more common procedures currently inuse include direct sequencing of target regions amplified by PCR™ andsingle-strand conformation polymorphism analysis (“SSCP”). SSCP reliesupon the differing mobilities of single-stranded nucleic acid moleculesof different sequence on gel electrophoresis. Techniques for SSCPanalysis are well known in the art.

Another method of screening for point mutations is based on RNasecleavage of base pair mismatches in RNA/DNA and RNA/RNA heteroduplexes.As used herein, the term “mismatch” is defined as a region of one ormore unpaired or mispaired nucleotides in a double-stranded RNA/RNA,RNA/DNA or DNA/DNA molecule. This definition thus includes mismatchesdue to insertion/deletion mutations, as well as single and multiple basepoint mutations.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Induction of Targeted Mutations Zinc Finger Design

A pair of ZFNs were designed and constructed for a chromosomal targetlocus in the yellow (y) gene of Drosophila. Zinc fingers generally bindpreferentially to G-rich regions of DNA, and extensive study has beenperformed of fingers that bind all 5′-GNN-3′ triplets (Segal et al.(1999) PNAS USA 96: 2758-2763). Because the binding sites must be in aninverted orientation with respect to each other for effective cleavageby ZFNs (Bibikova et al. (2001) Mol. Cell. Biol. 21: 289-297), thechromosomal target locus of Drosophila (y) was searched for invertedrecognition sequences of the form (NNC)₃ . . . (GNN)₃. Such a site wasidentified in exon 2 with a 6-bp separation between the component 9-merrecognition sites, which is the optimal spacer for specific recognitionand cleavage by ZFNs that have no added linker or spacer between thebinding and cleavage domains (M. Bibikova et al. (2001) Mol. Cell. Biol.21: 289-287). The specific recognition sequences of the two ZFNs aredescribed in Bibikova et al. 2002, Genetics 161: 1169-1175. DNAsencoding zinc fingers that recognize the DNA sequences, 5′-GCGGATGCG-3′(SEQ ID NO: 1) and 5′-GCGGTAGCG-3′ (SEQ ID NO: 2), were obtained fromDrs. David Segal and Carlos Barbas (Scripps Research Institute, LaJolla, Calif.) (Segal, D. J, et al. (1999) PNAS 96: 2758-2763). The DNAsencoding the zinc fingers were then modified using mutagenic PCRprimers, and two sets of three zinc fingers each were produced: one,referred to as yA that recognizes one of the component 9-mers of the ygene target (5′-GTG-GATGAG-3′ (SEQ ID NO: 3)), and another, referred toas yB, that recognizes the other component 9-mer of the y gene target(5′-GCGGTAGGC-3′ (SEQ ID NO: 4)). Two fingers were modified in yA, butonly one in yB. DNA encoding each of the resulting 3-finger sets of zincfingers were both cloned in frame with the FokI DNA cleavage domain inthe pET15b expression plasmid, with no intervening linker DNA betweenthe DNA recognition and cleavage domains. Both chimeric ZFN proteinswere expressed, purified by Ni-affinity chromatography, and tested forcleavage activity in vitro by methods described previously (Smith, J.,et al. (2000) Nucleic Acids Res. 28, 3361-3369; and Bibikova, M., et al.(2001) Mol. Cell. Biol. 21, 289-297), using the pS/G plasmid (Geyer, P.K. & Corces, V. G. (1987) Genes Dev. 1, 996-1004), which carries thecomplete y gene. Together the two ZFNs made a single double strandedbreak (DSB) at the expected site in a 10.7-kb plasmid DNA carrying the ygene.

P Element Vectors and Transformation of Fly Larvae.

The yA and yB ZFN coding sequences were then cloned separately behindthe Drosophila Hsp70 heat shock promoter by insertion of ZFN DNA betweenthe BamHI and SalI sites of a modified phsp70 plasmid (Petersen, R. B. &Lindquist, S. (1989) Cell. Regul. 1, 135-149). A fragment carrying theheat shock promoter and ZFN DNA sequences was excised by partial HindIIIand complete Apal digestion and cloned between these same endonucleasesites in the commercially available cloning vector, pBluescript. Afterverification of the sequence of the insert, it was excised by digestionwith NotI and inserted into the ry+P element vector pDM30 (Mismer, D. &Rubin, G. M. (1987) Genetics 116, 565-578). The resulting yA and yBplasmids were injected separately into v ry embryos, along with theP-transposase expression plasmid pπ25.1wc, and eclosing adults weremated to screen for ry+ germline transformants. The ry+ insertion wasmapped to a specific chromosome for multiple independent transformantswith each ZFN. Both balanced and homozygous stocks were created forseveral lines carrying yA and yB without viability problems in mostcases. Genes for the two ZFNs were brought together (as described in theExamples below) with appropriate crosses of mature flies, and theoffspring were heat shocked 4 days after the initiation of mating byimmersing the glass vials containing the flies in a water bath at 35°for one hour. As adults eclosed they were screened for evidence ofsomatic y mutations. Control vials from crosses involving each nucleaseseparately were subjected to the heat shock, and yA+yB flies that hadnot been heat shocked were also screened.

Recovery of Germline Mutants.

All flies emerging from the heat shock protocol and carrying both the yAand yB nucleases were mated to reveal potential germline mutations.Males were crossed with 2 or 3 attached-X [C(1)DX] females, and theresulting male offspring screened for yellow body color. Females werecrossed with 2 or 3 y (FM6) males, and the resulting offspring of bothgenders screened. Mutants were identified and all of them were malesthat had originated from male parents. These identified mutant maleoffspring were then crossed to C(1)DX females to produce additionalprogeny carrying the same mutation.

DNA Analysis.

The presence or absence of the target DNA was identified by DNAanalysis. Individual flies were homogenized in 100 μl of a 1:1 mixtureof phenol and grind, buffer (7 M urea, 2% SDS, 10 mM Tris, pH 8.0, 1 mMEDTA, 0.35 M NaCl) preheated to 60°. Each sample was extracted with 50pi of chloroform, the organic phase back-extracted with 100 μl of grindbuffer, and the combined aqueous phases re-extracted with 50 μl ofchloroform. DNA was precipitated with ethanol and re-dissolved in 20 μlof 10 mM Tris, pH 8.5. A 600-bp DNA fragment was amplified by PCR withprimers flanking the yA+yB recognition site. The primers were called YF2(5′ATTCCTTGTGTCCAAAATAATGAC-3′ (SEQ ID NO: 5)) and YR3(5′-AAAATAGGCATATGCATCATCGC3′ (SEQ ID NO: 6)) For the larger deletions,YR3 was used in combination with a more distant sequence, YF1(5′ATTTTG-TACATATGTTCTTAAGCAG-3′ (SEQ ID NO: 7)). Amplified fragmentswere recovered after gel electrophoresis, and DNA sequences weredetermined at the University of Utah DNA Sequencing Core Facility withan ABI3700 capillary sequencer and the YR3 primer.

Induction of Targeted y Mutations Resulting from Double Stranded Breaksand Nonhomologous End Joining

The levels of expression of yA induced at 37° were found, in severalindependent transformants, to be lethal when applied at larval andembryonic stages. Moderating the heat shock to 35° allowed survival of agood proportion of the yA-carrying flies. The yB ZFN did not affectviability at any temperature tested.

After individual flies carrying the yA and yB nucleases on the samechromosome were crossed and their progeny heat-shocked, offspringdemonstrating y mosaic, as well as germline mutations were observed inmale offspring. In males (except following DNA replication), only simplereligation or NHEJ would be available to repair the damage after a DSB.In Drosophila, as in many other eukaryotes, NHEJ frequently producesdeletions and/or insertions at the joining site. Since the DSB istargeted to protein coding sequences in y+, most such alterations wouldlead to frame-shifts or to deletion of essential codons, which can leadto a phenotype of patches of y mutant tissue.

Somatic yellow mosaics were identified in multiple yA+yB males. Most ofthe patches were in the distal abdominal cuticle and bristles, but someexamples in leg, wing and scutellar bristles were also observed. Noother phenotypic defects have been seen on a regular basis. Thefrequency of somatic mosaics was quite high. In pooled data from crossesinvolving a number of independent yA and yB lines, 105 of 228 candidatemales (46%) showed obvious y patches. For some yA+yB combinations thefrequency was greater than 80%. No yellow mosaics were observed incontrols with a single nuclease or without heat shock. This indicatesthat the yA+yB ZFNs are capable of inducing somatic mutations at theirdesignated target.

Characterization of Germline y Mutations.

To isolate germline y mutations, all yA+yB males from several heat shockexperiments were crossed to females carrying an attached-X chromosome[C(1)DX/Y], in order to produce male offspring that were known to onlyreceive their father's X chromosome. In total, 228 male fathers yielded5,870 sons; 26 of the male off-spring, from 13 different fathers, wereclearly y throughout their entire bodies. Thus, 5.7% of the yA+yB malefathers produced at least one germline mutant. Of the 13 fathers, 6 hadbeen identified as having y somatic patches, while the other 7 appearedto be entirely y+in diagnostic features. No y flies were isolated among7050 progeny of 125 heat-shocked yA+yB females crossed to y males. TheZFNs appear to be effective in inducing mutations via NHEJ mostefficiently in the male germline.

DNA was isolated from the 13 fathers identified above and 5 additionalmales in order to analyze each of them for the presence of the targetDNA. A 600-bp fragment including the expected cleavage site wasamplified by PCR. In three of the 18 male flies, the binding site forone of the primers had been deleted, and a new primer had to begenerated in order to accomplish amplification. This new primer waslocated at a more distant location. Sequence analysis of all fragmentsrevealed unique alterations precisely at the target site. Nine of thesequenced mutants had simple deletions; five had deletions accompaniedby insertions; and three were simple, short duplications. Three of thedeletions extended for hundreds of bps to one side of the target andthese were the three samples that required a new primer design. Theseare exactly the types of mutations that were expected to result fromNHEJ after cleavage by the yA+yB ZFNs, and they are very similar tothose produced after P element excision. Some of the frameshift ymutations created a stop codon within a short distance of thealteration, while one inserted an asparagine codon into the normalreading frame.

Targeted Cleavage and Mutagenesis.

This example demonstrated that ZFNs can be designed to produce DSBs intarget chromosomal locus in an exemplary genome in order to produce apermanent genetic alteration. The frequency of observed somatic mutationwas quite high, and the real number of somatic mosaics may be evenhigher, since y mutations have no effect on many visible features. Thiswas corroborated by the recovery of germline mutations fromphenotypically y+ parents.

In this particular Example, germline mutations were recovered only inmales and at a lower frequency than somatic mosaics.

Example 2 ZFN-Induced Double Stranded Breaks Stimulate Targeted GeneticRecombination in the Presence of Homologous Donor DNA Zinc Finger andDonor DNA Design

A pair of ZFNs were designed and constructed for a chromosomal targetlocus in the yellow (y) gene of Drosophila as described in Example 1.

In order to make an identifiable donor DNA for the Drosophila gene, y,the yA and yB recognition sites for the zinc fingers were replaced withtwo in-frame stop codons and an XhoI site. These changes were introducedby amplification with PCR primers carrying the desired sequence.Relative to the wild type y, 21 bp were deleted leaving only 3 bp of theyA recognition site, and a 9 bp replacement inserted the two in-framestop codons and inserted the XhoI site. This mutant (yM) carries a totalof 8 kb of homology to the y locus. It was inserted into a P elementvector and introduced into the fly genome. The yM sequence is flanked byrecognition sites for the FLP recombinase (FRT) and the meganucleaseI-SceI to permit excision and linearization of the donor. Generating alinear extrachromosomal donor DNA in situ by this means has been shownto enhance its effectiveness in recombination (Y. S. Rong and K. G.Golic, Science 288, 2013-2018 (2000)).

Experimental Design

The design of the targeted genetic recombination experiment is asfollows: The y⁺ target lies on the X chromosome. The transgenes for theyA and yB ZFNs are on one chromosome 2, while those for FLP and/orI-SceI (when present) are on the other chromosome 2. The donor DNA (yM)is located on chromosome 3 in a p-element vector that also carries thewhite gene (W⁺). Each of these inserted genes is under the control of aDrosophila HSP70 promoter. Upon heat-shock induction, the ZFNs will cuttheir target at y. This broken chromosome can be restored to wild type,or it can acquire a y mutation either by NHEJ or by homologousrecombination. When neither FLP nor I-SceI is present, the donor remainsintegrated. When FLP is expressed, the donor is excised as anextrachromosomal circle. When I-SceI is also expressed, it converts thedonor to an ends-out linear molecule which can recombine with thecleaved target locus. Experiments were also performed with linear donoronly in the absence of yA and yB (and therefor without cleavage of thetarget).

Larvae carrying single copies of these introduced DNA components wereheat-shocked at 35°, for one hour, 0-4 days after egg laying. Theexperiment contained five groups as exemplified below:

ND, no donor: yA+yB only;

ID, integrated donor: yA+yB+donor, no FLP or I-SceI;

CD, circular extrachromosomal donor: yA+yB+FLP+donor;

LD, linear extrachromosomal donor: yA+yB+FLP+I-SceI+donor;

DO, linear donor only: FLP+I-SceI+donor, but no ZFNs.

Adults emerging from the heat shock protocol were crossed to revealgermline y mutations. The frequencies of germline y mutations resultingfrom the heat-shock treatment are shown in Table 1 in column 3. Thefrequencies of mutation rose in both males and females in the presenceof the donor and the frequency increased further with extrachromosomaland linear DNA. With linear extrachromosomal DNA, nearly 20% of malesand 14% of females yielded at least one mutant offspring.

The y mutations were propagated in further crosses, chromosomal DNA wasrecovered. The frequency of germline y mutants and the proportion due toeither NHEJ or homologous recombination with the donor DNA wasdetermined by PCR amplification of 600 bp of DNA including the targetregion of the y gene followed by XhoI digestion of the amplifiedproduct. Products of homologous recombination between donor and targetwere recognized by XhoI digestion of the PCR fragment; some of these andmany of the XhoI-resistant products were sequenced. The latter showedsmall deletions and/or insertions and occasionally larger deletions, allof which are characteristic of NHEJ.

The fourth column of Table 1 reports the recovery of germline mutants asa percentage of all offspring. The fractions of those mutationsresulting from either NHEJ or homologous recombination with the donorrose as the donor DNA became more effective at participating inhomologous recombination: linear donor DNA being more effective thancircular donor DNA, which was more effective than integrated donor DNA.The integrated donor, located on chromosome 3, was not very effective inserving as a template for repair of the break at y and the majority ofrecovered mutations were due to NHEJ. The circular donor was much moreeffective and approximately ⅓ of all mutations were determined to be dueto gene replacements. With the linear donor, more than 2% of all sons ofmales were mutant, and 63% of these were products of homologousrecombination. In the female germline 73% of y mutations were homologousreplacements. Target cleavage by chimeric ZFNs stimulates targetedgenetic recombination substantially, and the most effective way tointegrate donor DNA into a host organism's genome is with linear donorDNA.

The ZFN-induced targeted genetic recombination results differ from thoseobtained without targeted cleavage in several respects. First, inducedmutations were found in both the male and female germlines, while onlyfemales had yielded good frequencies in previous trials by otherresearchers. Apparently the presence of a DSB in the target activatesrecombination processes in males that are not efficient on intactchromosomes. The lower targeting frequencies observed in females mayreflect the possibility of repairing the break by recombination with anuncut homologous X chromosome. Second, the overall frequency of inducedmutations was about 10-fold higher in males in the linear DNA andcircular DNA experiments than was seen earlier at y in females with anends-in donor: approximately 1/50 gametes, compared to 1/500 gametes.Even in the female germline, the frequency of ZFN-induced mutations was1/200 gametes, and ¾ of these were gene replacements. Thus, the presenceof a homologue donor does not preclude interaction with theextrachromosomal donor. Third, deletions and insertions due to NHEJ werealso observed, in addition to the targeted homologous recombinants. Suchproducts were not expected nor observed in the absence of targetcleavage.

Example 3 Expression of Chimeric ZFNs in Arabidopsis in Order toStimulate Induction of Targeted Mutations

Experimental Design The method of the present invention will be used totarget and knock out the Arabidopsis TRANSPARENT TESTA GLABRA1 gene(TTG1, gene number AT5G24520 (GenBank number AJ133743). An EST for thisgene has been sequenced (GenBank numbers F20055, F20056). The geneencodes a protein containing WD40 repeats (Walker et al. (1999) PlantCell 11, 1337-1349).

Two chimeric DNA constructs will be generated consisting of (1) nucleicacid sequence encoding the promoter region from the Arabidopsis HSP18. 2gene and (2) nucleic acid sequence encoding zinc finger proteinsspecific for the TTG1 gene operatively linked to a nucleic acid sequenceencoding a non-specific endonuclease. The HSP18.2 promoter will conferexpression in Arabidopsis and gene expression will be controlled byheat-shocking the resulting plants. The chimeric genes will be referredto as HS::ZnTTG1 A and HS::ZnTTG1B. These two genes can be incorporatedinto the same Agrobacterium vector.

All of our experiments will be carried out using the model geneticorganism Arabidopsis thaliana, because of a number of desirable featuresof this system including small size, small genome, and fast growth. Attg1 mutant has a distinctive phenotype, making it an excellentexemplary model. For instance, ttg1 mutants are glabrous and mutantplants lack trichomes on leaves and stems. Trichomes are hair-likeoutgrowths from the epidermis.

Additionally, ttg1 mutant are defective in flavonoid production.Flavonoids are a complex class of compounds including purple anthocyaninpigments and tannins TTG1 protein positively regulates synthesis of theenzyme dihydroflavonol reductase, which is required for production ofboth anthocyanins and tannins (Shirley et al. (1995) Plant Journal 8:659-671; Pelletier and Shirley (1996) Plant Physiology 111: 339-345).

These ttg1 mutants also have a transparent testa or seed coat. In wildtype, the seed coat (inner layer of the inner integument) has dense,brown tannin and ttg1 mutants lack this pigment. As a consequence, theseed coat of seed collected from ttg1 mutants are transparent, and seedcollected from ttg1 mutants are yellow because the yellow embryos showthrough the transparent seed coat.

These ttg1 mutants also lack anthocyanins. In wild type, seedlings,stems, and leaves produce reddish/purple anthocyanin pigments,particularly under stress. These pigments are absent in ttg1 mutants.

Additionally, ttg1 mutants produce extra root hairs. In wild type, roothairs are produced only from trichoblast cells. In ttg1 mutants, bycontrast, root hairs are produced by both trichoblast cells andatrichoblast cells. The result is a root that appears more hairy (Galwayet al. (1994) Developmental Biology 166, 740-754).

The ttg1 mutants also fail to produce mucilage in the outer layer of theseed coat. Mucilage is a complex carbohydrate, sometimes called slimethat covers the seed coat. Lastly, the ttg1 mutants have altereddormancy and ttg1 seeds do not require drying out or cold treatments togerminate.

The presence of all seven characteristics makes visual screening forthis mutant genotype an easy task.

Design of Zinc Fingers

The TTG1 gene was scanned for sequences of the form: NNY NNY NNY RNN RNNRNN, where Y is either T or C, R is A or G, and N is any base. Thisidentified sequences comprised of triplets that are initiated by an A orG in opposite orientation—i.e., on opposite strands—and separated byexactly 6 bp. This has been shown to be a preferred structure for zincfinger nuclease recognition and cleavage (M. Bibikova et al. (2001) Mol.Cell. Biol. 21: 289-287).

The component triplets of the sequences identified in 1 were thenclassified according to whether there were zinc fingers that were knownto bind them specifically. Two sites in TTG1 were identified aspotential ZFN binding and cleavage sites: 5′-TCC GGT CAC AGA ATC GCC GTCGGA-3′ (SEQ ID NO: 8), and 5′-ACT TCC TTC GAT TGG AAC GAT GTA3′ (SEQ IDNO: 9) (at nucleotide 406 in the TTG1 sequence).

Zinc finger nucleases comprising a binding domain designed to bind thefirst of these sites will be constructed either by oligonucleotidesynthesis and extension (Segal, D. J. (2002) Methods 26: 76-83), or byPCR with mutagenic primers (M. Bibikova et al. (2002) Genetics161:1169-1175). The resulting coding sequences will be inserted intoplasmids vectors in frame with the FokI nuclease domain to create twoZFN coding sequences, ZnTTG1A and ZnTTG1B. The encoded proteins will beexpressed in E. coli and partially purified(M. Bibikova et al. (2002)Genetics 161:1169-1175). The recovered ZFNs will be tested in vitro forthe ability to cleave plasmid DNA encoding the TTG1 gene. Success inthis assay will be evidenced by no cleavage by either ZFN alone, butcleavage at the expected site by a mixture of the two ZFNs.Transformation:

The HS::ZnTTG1A and HS::ZnTTG1B genes will be introduced into theArabidopsis genome using Agrobacterium-mediated transformation. To doso, the HS::ZnTTG1A and B genes will be inserted into an AgrobacteriumT-DNA transformation vector (pCAMBIA1380) that harbors a selectablehygromycin resistant marker. A pCAMBIA HS::ZnTTG1 clone will then beintroduced into Agrobacterium cells using standard Agrobacteriumtransformation procedures, and the HS::ZnTTG1A and HS::ZnTTG1B geneswill then be introduced into Arabidopsis plants using the standardfloral dip method. (See, Clough, S. and Bent, A (1999) Plant Journal 16:735-743).

Induction of Expression of ZFNs in a Host Cell

Seeds from the T1 generation will be collected from the dipped plants.In order to select for transformed seedlings, the T1 seeds will begerminated on agar plates containing the antibiotic hygromycin.Approximately four days after germination, the plates containing thegerminated seedlings will be wrapped in plastic wrap and immersed in 40°C. water for two hours to induce expression of the ZFN genes. Atapproximately two weeks following germination, the hygromycin resistanttransformed seedlings will be transferred to dirt.

Screening for Gene-Targeting Event:

Screening Method 1: The HS::ZnTTG1 genes will be introduced intowild-type Arabidopsis plants and the T1 plants will be heated asdescribed above. At 1-2 weeks following heat treatment, a sample oftissue will be harvested from heat-treated plants and DNA extracted fromthis tissue. PCR amplification using 20 bp primers flanking the zincfinger target site (25 bp on each side of the target site) will beutilized to determine if the HS::ZnTTG1 gene is present. The PCR bandfrom control plants that were not heat treated should be approximately90 bp in size. PCR bands from the heat-treated plants should includesmaller products than 90 bp that result from the existence of deletionssurrounding the zinc finger target site. To verify the existence ofsmall deletions, we will clone and determine the DNA sequence of thesmaller PCR products.

Screening Method 2: The HS::ZnTTG1 A and HS::ZnTTG1 B genes will beintroduced into wild-type. Arabidopsis plants and the T1 plants will beheat-treated as described above. The T1 plants will be grown tomaturity, allowed to self pollinate, and T2 seeds will be collected. TheT2 seeds will be grown on agar plates and they will be scored forseedling phenotypes including hairless leaves (glabrous phenotype),brighter leaves (anthrocyanin minus phenotype), and hairy roots, asdescribed above. Mutant plants will be transferred to dirt and grownfurther. Tissue from mutant plants will be harvested and DNA extractedin preparation for PCR—screening as described above. Briefly, PCR willbe performed with primers flanking the zinc finger target sites andsamples exhibiting approximately 90 bp products were not transformed,whereas those exhibiting products less than 90 bp were transformed. Thisis due to the existence of deletions surrounding the zinc finger targetsite. Additionally, small insertions or much larger deletions may bepresent around the zinc finger target site, as well. To verify theexistence of these occurrences, we will clone and determine the DNAsequence of the smaller PCR products.

Screening Method 3:

The HS::ZnTTG1 A and HS::ZnTTG1 B genes will be introduced intoheterozygous ttg1 mutants (i.e., genotype ttg1/TTG1). The male sterilel(ms1) plants will be introduced to the Agrobacterium solution (note: thems1 and ttg1 loci are linked, 6 cM apart on chromosome 5). The dippedplants then will be pollinated with pollen from homozygous ttg1-1plants. The crossed plants will be allowed to mature, the resultantT1/F1 seeds collected, and the T1/F1 seeds allowed to germinate in thepresence of hygromycin. Surviving T1/F1 seedlings will contain theHS::ZnTTG1 transgene and will be heterozygous at the ttg1 locus (i.e.,genotype MSI-ttg1-1/ms1-TTG1). The T1/F1 plants will be heat-shocked asdescribed above. In a subset of cells, the wild-type allele will beknocked out, resulting in a sector of homozygous ttg1 (i.e., genotypettg1-1/ttg1-ko) cells. These mutant sectors will be detectable (and,thus, a targeted genetic recombination event) by visualizing severalphenotypes, such as hairless leaves (glabrous phenotype), brighterleaves (anthocyanin minus phenotype), and yellow seeds (transparenttesta phenotype). Tissue will be collected from mutant sectors andtargeting verified using the PCR-cloning-sequencing strategy discussedabove. From the mutant sectors, T2 seeds will be collected and growninto T2 plants. In the T2 generation, the phenotype will be verified:plants homozygous for the knockout allele (i.e., ttg1-ko) also will behomozygous for the ms1 mutation and, thus, will be male sterile (i.e.,genotype ms1-ttg1-ko/ms1-ttg1-ko). Tissue from the double mutants(phenotypically ttg1 and ms1) will be harvested and verified fortargeting using the PCR-cloning-sequencing strategy discussed above.

All of the COMPOSITIONS, METHODS and APPARATUS disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the COMPOSITIONS, METHODS and APPARATUS and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itwill be apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

TABLE 1 Recovery of Germline y mutations 1 2 3 4 Donor # Screened #Giving y Total y Females: ND 125 0 (0%) 0 ID 188   9 (4.8%) 15 (0.16%)CD 309 31 (10%) 59 (0.38%) LD 503   68 (13.5%) 135 (0.54%)  DO 158   1(0.6%)  2 (0.02%) Males: ND 228  13 (5.7%) 24 (0.42%) ID 218 24 (11%) 40(0.73%) CD 261 49 (19%) 104 (1.59%)  LD 522 94 (18%) 292 (2.24%)  DO 177  1 (0.6%)  1 (0.02%)

1.-98. (canceled)
 99. A plant cell comprising a mutation introduced by aZinc Finger Nuclease (ZFN) that binds to a target site in a chosen hostchromosomal target locus; wherein the mutation comprises an insertionand/or deletion of genetic material surrounding the target site. 100.The plant cell of claim 99, wherein the mutation is an insertion ofgenetic material.
 101. The plant cell of claim 99, wherein the mutationis a deletion of genetic material.
 102. The plant cell of claim 99,wherein the mutation is both a deletion and an insertion of geneticmaterial.
 103. The plant cell of claim 99, wherein a donor DNA isinserted into the chromosomal target locus.
 104. The plant cell of claim103, wherein the donor DNA provides a gene sequence that encodes aproduct to be produced in the plant cell.
 105. The plant cell of claim103, wherein the donor DNA provides a gene sequence that encodes apharmaceutical, hormone, protein, nutriceutical or chemical.
 106. Theplant cell of claim 99, wherein the method further comprises: selectinga zinc finger nuclease comprises a zinc finger protein DNA bindingdomain capable and a non-specific DNA cleavage domain capable ofcleaving double-stranded DNA when operatively linked to said DNA-bindingdomain.
 107. The plant cell of claim 106, wherein the DNA binding domainis comprised of three zinc fingers.
 108. The plant cell of claim 106,wherein the zinc finger DNA binding domain is a Cis2His2 zinc finger.109. The plant cell of claim 106, wherein the cleavage domain is from aType II restriction endonuclease.
 110. The plant cell of claim 109,wherein the Type II restriction endonuclease is Fold.
 111. The plantcell of claim 104, wherein the gene sequences encodes one or moreselectable markers.
 112. The plant cell of claim 111, wherein the one ormore selectable markers provides positive selection for cells expressingthe marker.
 113. The plant cell of claim 111, wherein the one or moreselectable markers provides negative selection for cells expressing themarker.
 114. The plant cell of claim 111, wherein the selectable markerprovides positive and negative selection for cells expressing themarker.
 115. The plant cell of claim 104, wherein the donor DNAcomprises a constitutively active or inducible promoter upstream of thegene sequence that encodes a product to be produced in the plant cell.